Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haematopoietic growth factor which in vitro stimulates the survival, proliferation, differentiation and function of myeloid cells and their precursors, particularly neutrophil and eosinophil granulocytes and monocytel/macrophages (for review, see 1). The in vivo effects of GM-CSF have been studied in murine models by infecting pharmacological doses of GM-CSF (2), by generating GM-CSF tranagenic mice (3), and by reconstituting lethally irradiated mice with bone marrow cells overproducing GM-CSF (4).
These studies confirm the haematopoietic activity of GM-CSF in vivo, and suggest that excess levels of GM-CSF may be implicated in some disease processes. However, the usual physiological role of GM-CSF is not well defined (5). Endogenously-produced GM-CSF is not usually detectable in serum (6), and in humans altered serum levels have not correlated clearly with haematological or disease processes (1,6). It has been suggested that GM-CSF may be produced and act locally (1), but the cells producing G-CSF in vivo have yet to be identified. Moreover, it is not clear whether GM-CSF is an essential regulator for steady-state production of granulocytes and macrophages, or whether it is required as a regulator for emergency haematopoiesis in response to challenges such as bacterial infection. It is also not known whether GM-CSF is involved in the normal development of non-haematopoietic tissues.
Granulocyte colony-stimulating factor (G-CSF) is a haematopoietic growth factor which i vitro controls granulopoiesis. It stimulates the survival, proliferation, differentiation and function primarily of neutrophil granulocytes. As in the case of GM-CSF, the in vivo effects of G-CSF have been studied in murine models by injection of pharmacological doses of G-CSF, by generation of GM-CSF transgenic mice, and by reconstitution of lethally irradiated mice with bone marrow cells transformed with a retroviral vector carrying cDNA encoding G-CSF (reviewed in Reference 7). However, as with GM-CSF, the usual physiological role in vivo of G-CSF is unclear. It may act as a regulator in steady-state granulopoiesis, or may function as a regulator for emergency granulopoiesis in response to specific challenges requiring increased neutrophil production, such as infection (7).
G-CSF and its isolation, characterisation, and recombinant production have been extensively reviewed, for example papers cited in Reference 1.
Until recently, genetic studies depended upon the discovery of random mutations (either spontaneous or induced) or of pre-existing genetic polymorphisms. However, following the rapid development of recombinant DNA technology and of identification of specific genes, particularly in mice, by analogy to genes from other species or from the biochemistry of the protein products which they encode, methods for specifically-targeted deletion or modification of genes have been developed. Provided that a cloned, genomic fragment of the chosen genetic locus is available, it is possible to generate null alleles by disruption of the gene, to modify functional properties of the gene such as transcriptional pattern, mRNA or protein maturation pattern, or to modify the ability of the protein to interact with other gene products. This is achieved by using conventional recombinant DNA methods to introduce the desired mutation into a cloned DNA sequence of the chosen gene; the mutation is then transferred by means of homologous recombination into the genome of a pluripotent embryonic stem cell (ES cell). The ES cells thus produced are transferred by microinjection into mouse blastocysts in order to generate germ-line chimeras. Animals homozygous for the desired mutation are then generated by interbreeding of heterozygous siblings.
These techniques are now widely used, and have been employed to generate lineages of mice in which a variety of genes are disrupted; these mice are often referred to as "knock-out" mice. Many of these mutations are lethal, causing death early in embryonic life or in the perinatal period. The technique has been most successful in producing "knock-out" mice in which genes for molecules of immunological importance or for growth factors are deleted. The techniques are well established, and a variety of marker genes and genes employed to assist in selection of cells which have undergone homologous recombination rather than random integration of DNA are available. A number of reviews have been published [7 to 11], and techniques for generation of transgenic mice in general are reviewed in International Patent Application No. WO 91/13150 by Ludwig Institute for Cancer Research.
While such gene targeting is useful in the production of mouse models for genetically-determined human diseases, which models can be used for testing potential therapies, and while the techniques are well established in principle, it is not possible to predict in advance whether an animal line bearing a given targeted gene disruption can be generated, or if so how readily practicable generation of such a model will be and the best experimental approach to utilise. In particular, the frequency of transformation of the ES cells varies widely, from as little as 1 in 40,000 to as much as 1 in 150. While some of the factors involved in optimisation of transformation frequency are known, success is not easy to predict.
We have generated GM-CSF deficient and G-CSF deficient mice through targeted disruption of the GM-CSF and G-CSF genes respectively in embryonic stem cells. We have surprisingly found that while GM-CSF deficient mice have no major perturbation of haematopoiesis, they all have abnormal lungs and are prone to lung infections, implicating GM-CSF as being essential for normal pulmonary physiology and resistance to local infection. GM-CSF deficient mice are useful as a model system for the syndrome of alveolar proteinosis, and are particularly useful as a model system for the study of opportunistic infections and infections which are intractable to currently available therapies.
G-CSF deficient mice are neutropaenic, but have normal levels of monocytes/macrophages in the periphery. They are prone to sub-clinical infections, and will be useful in testing the efficacy of anti-microbial agents, especially in settings of increased vulnerability. They will also be useful in assessing the virulence of microorganisms.